- Email: [email protected]
Where is supercritical fluid extraction going?
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Where is supercritical £uid extraction going? M.D. Luque de Castro*, M.M. Jimeènez-Carmona
Analytical Chemistry Division, Faculty of Sciences, E-14004 Coèrdoba, Spain The status of supercritical £uid extraction as an analytical technique for treatment of solid samples is discussed. Its most signi¢cant advantages are its preconcentration effect, cleanness and safety, quantitativeness, expeditiousness and simplicity. Its limitations include the dif¢culty of extracting polar analytes, owing to the non-polar character of the CO2 which is used, the different recoveries obtained from spiked and natural samples, and the frequent need for clean-up steps after extraction. A critical discussion is given of these factors, as well as possible solutions and new alternatives such as the use of sub- and supercritical water and accelerated solvent extraction. z2000 Elsevier Science B.V. All rights reserved. Keywords: Analytical supercritical £uid extraction; Sample treatment; Polar analyte
1. Introduction The continued search for rapid, ef¢cient and cost-effective means of sample treatment has introduced supercritical £uids into analytical chemistry, giving us supercritical £uid chromatography (SFC ) and supercritical £uid extraction (SFE ) [ 1 ]. Both techniques seek to exploit the unique properties of gases at temperatures and pressures above the critical point. SFE is a somewhat atypical technique in its chronological evolution. Its chromatographic counterpart, SFC, was developed at an earlier stage ( ¢rst demonstrated in 1962 [ 2 ]), which is in clear contrast with other extraction techniques whose batch modes were developed much earlier than their continuous modes ( e.g., liquid^liquid extraction ). On the other hand, the fairly extensive commercial availability of supercritical £uid extractors when the technique was introduced was inconsistent with the relative scarcity of associated publications and developments, as compared to most analytical *Corresponding author. Fax: +34 (957) 218 606. E-mail: [email protected]
techniques. Traditionally, the manufacture of commercially available apparatus and instruments, which has fostered the development of new applications and their customary use, has followed scienti¢c and technical advances and feasibility tests. These seemingly contradictory facts are easy to explain, though, in that SFE aids ef¢cient development of the ¢rst stage of the analytical process, which is also the most needed in research. The SFE literature shows the predominant use of CO2 as extractant ( more than 98% of the applications have been developed using this £uid ) and calls for an inspection of its features to show whether the decreased research in this ¢eld is related to the extractant itself. In the authors' opinion, supercritical CO2 has signi¢cant advantages, but also shows important limitations that can be overcome with the help of new techniques and analytical approaches. An ideal extraction method should be rapid, simple, and inexpensive to perform. It should yield quantitative recovery of the target analytes without loss or degradation; provide a sample that is immediately ready for analysis without additional concentration or class fractionation steps; and generate no additional laboratory wastes. Unfortunately, liquid solvent extractions frequently fail to meet these goals. They often require several hours ^ or even days ^ to perform, result in a dilute extract ( which must be concentrated when trace analysis is required ), and may not result in quantitative recovery of the target analytes. Recent concerns about the hazardous nature of many commonly used solvents, the costs and environmental dangers of waste solvent disposal, and the emission of hazardous solvents into the atmosphere, have led to the development of alternative extraction methods. In this context, SFE ^ the technique based on the use of supercritical £uids as leaching agents ^ emerged in the mid-1980s as a promising tool to overcome the dif¢culties of solid sample extraction. The majority of analytical supercritical £uid extractions have used supercritical CO2 as extracting agent because of its preferred critical properties ( critical temperature and pressure, 31³C and 72 bar, respec-
0165-9936/00/$ ^ see front matter PII: S 0 1 6 5 - 9 9 3 6 ( 9 9 ) 0 0 2 2 8 - 9
ß 2000 Elsevier Science B.V. All rights reserved.
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tively ), low toxicity, and chemical inertness. Despite the promising features of this technique, it has not ful¢lled the expectations of researchers so far. Thus, many analysts were quick to try the technique but became frustrated and gave up SFE systems that did not live up to their expectations, leading to a reduction in the number of SFE instrument manufacturers. Here we review critically the current state of SFE in analytical chemistry, considering all aspects of the technique, as well as solutions to the drawbacks, and the foreseeable trends. Other reviews on this issue have been published recently, referring to general aspects of the technique [ 3 ], and speci¢c matters such as the use of SFE in food analysis [ 4,5 ], in the extraction of metals as complexes [ 6 ], or in the extraction of pharmaceuticals [ 7 ].
2. Advantages of SFE The bene¢ts of SFE ( focused on the use of supercritical CO2 , since this is mainly used ), versus conventional techniques which are based on the rapid diffusion of the analytes in the £uid ( gas-like diffusion ) and the £uid solvation power ( liquid-like solvation ), have led to SFE being proposed as an alternative to conventional liquid solvent extractions such as Soxhlet and ultrasonic extractions. The most signi¢cant advantages of the SFE technique are the preconcentration effect it has, its cleanness and safety, its quantitativeness, expeditiousness, and its simplicity and selectivity.
2.1. Preconcentration effect The preconcentration effect allowed by CO2 -SFE is one of its most signi¢cant advantages versus other extraction techniques, since the large solvent volumes used in Soxhlet extractions ( 50^200 ml ) must be concentrated to 2^5 ml by using an appropriate device prior to analysis in order to obtain adequate analyte concentrations. This step usually takes between 2 and 24 h, and releases substantial amounts of solvents into the atmosphere. By contrast, SF extracts are usually collected in smaller volumes ( 10^20 ml maximum ), thus avoiding the preconcentration steps required after other conventional techniques which are commonly used for sample extraction or digestion. The solvent changeover occasionally required for the isolation or determination of analytes is
most readily done in SFE. After extraction, the SF is separated by depressurisation and the analytes are collected by bubbling them through a suitable solvent, or deposited on a sorbent or impact solid, and subsequently dissolved in the most appropriate solvent for the intended purpose.
2.2. Cleanness and safety Extraction by SC-CO2 is cleaner and less hazardous than conventional extraction since CO2 is nontoxic and non-£ammable and, unlike toxic solvents ( n-hexane, ether, methylene chloride ), carbon dioxide poses no ¢re risk and leaves no environmentally hazardous wastes.
2.3. Quantitativeness Conventional extraction methods do not necessarily rely on complete isolation of the analyte, so increasing the recovery of an analyte, or extracting it quantitatively, detracts from the extraction's precision and reliability. The SFE extraction of 2,3,7,8tetrachlorodibenzo-p-dioxin from sediments [ 8 ] is a good example of how quantitative the extraction with SFs can be. Virtually 100% of the analyte was extracted within 30 min from the sediment, whilst Soxhlet extraction of the same sample for 18 h provided only 65% recovery. The recovery achieved by SFE ( under optimum working conditions ) can sometimes be even higher than that provided by Soxhlet. This is because the energy provided by solvents in Soxhlet extraction is not high enough to break the very strong bindings that sometimes occur with natural samples, and can necessitate the use of more drastic conditions such as those provided by SFE. This is the case with the extraction of hydrocarbons [ 9 ] or nitroaromatic and polyaromatic compounds [ 10 ] from soils. The solvent power, polarity, and working temperatures of the SFE, as well as its high diffusivity, are the chief factors on which complete extraction relies.
2.4. Expeditiousness One of the most valuable properties of SFE relative to classic separation techniques is expeditiousness. In fact, it is faster than conventional liquid extraction since: ( a ) the SFE penetrates into solid matrices more rapidly than do liquid solvents; ( b ) no concentration is required after extraction; and ( c ) the time needed to make the sample ready for
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measurement is up to 100 times shorter in many cases. Thus, CO2 extraction at 300 atm during 5 min gave recoveries of diclofol residues from ¢sh slightly higher ( 94.1^99.8%) than those provided by Soxhlet extraction with hexane during 8 h ( 87.9^92.3%) [ 11 ].
2.5. Simplicity SFE involves a very small number of steps, so it leads to short analysis times and reduced sample transfer, and hence to small analytical errors. The simplicity of SFE is clearly re£ected in the extraction of atrazine from soil samples. Supercritical CO2 extraction of a soil sample for 15 min with no previous treatment is enough to obtain recoveries comparable to those provided by Soxhlet extraction after sample pretreatment.
2.6. Selectivity One of the most important advantages of SFE versus conventional extraction techniques is its selectivity. Thus, SFE allows selective extraction of a wide range of analytes (PAHs, PCBs, and organochlorides ) [ 12 ] while not extracting the bulk matrix ( e.g., humic acids ). This behaviour allows the extract to be ready for direct analysis without additional manipulation.
3. Limitations of SFE Supercritical CO2 extraction, whose most important bene¢ts have been described, also shows signi¢cant limitations that are worth studying in order to know the ¢eld of application of the technique and help one seek for alternatives in this context. The main drawbacks SFE has to cope with are the dif¢culties in extracting polar analytes, the different ef¢ciencies obtained from spiked and natural samples, and the need for a clean-up step prior to measurement.
3.1. Dif¢culties in extracting polar analytes As stated above, the most popular £uid for SFE has been CO2 because of its low critical properties, low toxicity, and chemical inertness. Although CO2 is an excellent solvent for non-polar analytes, its main shortcoming is its low dielectric constant, which makes the extraction of polar and ionic com-
pounds dif¢cult. As examples, analytes such as alcohol phenol ethoxylate [ 13 ] and clenbuterol [ 14 ] are not extracted at all when pure CO2 is used as extractant. The strategies used in this context for facilitating the SFE of polar and ionic compounds have been reviewed recently [ 15 ] and are based on two general principles. The ¢rst strategy is to increase the polarity of the CO2 used as extractant, by changing the physical parameters of the supercritical CO2 , or by using supercritical £uids other than CO2 . The ¢rst choice would be a £uid with higher solvent strength, but the use of more polar £uids is often limited by practical considerations. Supercritical ammonia would be very attractive from a solvent strength standpoint, but it is dif¢cult to pump ( it tends to dissolve pump seals ), is chemically reactive, and is too dangerous for routine use. Supercritical methanol is also an excellent solvent but is less attractive because of its high critical temperature and because it is a liquid under ambient conditions, which complicates sample concentration after extraction. One of the most promising alternatives in this context is sub- or supercritical water, whose potential for extraction has been reviewed recently [ 16 ]. The most outstanding feature of this leaching agent is the possibility of manipulating its dielectric constant, making possible a drastic reduction in this parameter by raising the temperature under moderate pressure. For example, the dielectric constant of water decreases from 80 at ambient pressure and temperature, to 27 at 250³C and pressure s 40 atm, to a value below 10 under supercritical conditions. Thus, water has been used successfully to extract from environmental samples pollutants with a wide range of polarities, by sequentially raising the temperature [ 17,18 ], as well as for the extraction of pesticides from soils [ 19 ] and of essential oils from natural products [ 20,21 ]. Another way of increasing the polarity of the CO2 is by the addition of co-solvents, mainly methanol [ 22 ]. Modi¢ers can be introduced as mixed £uids in the pumping system, or with the aid of a second pump, or by simply injecting the modi¢er as a liquid onto the sample before beginning the extraction. A second group of strategies used for enhancing the SFE of polar and ionic compounds is based on reducing the polarity of the analyte to be extracted, by ion pair formation [ 23 ], esteri¢cation, or analogous reactions [ 24 ], organometallic compound formation [ 25 ], complex formation [ 26 ], or reverse micelle formation [ 27 ]. All these strategies compli-
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cate the experimental set-up and / or sample handling.
3.2. Dif¢culties in dealing with natural samples The samples typically subjected to SFE are solids, although the starting sample can be also semi-solid, liquid or gaseous. When the sample is not solid, it must be retained on an appropriate support ( a natural matrix from which the target analytes are removed previously, or a synthetic support such as glass wool or a diatomaceous earth ) in order to ensure effective attack by the supercritical £uid. It is also worth noting that most research work on SFE has been conducted on spiked samples. One of the most signi¢cant limitations of the technique in this context is that comparative studies involving natural and spiked samples have shown the former to deviate from the foreseeable behaviour in that the analytes bind more strongly in natural samples, which makes unfeasible the prediction of the behaviour of a natural sample from the results provided by a spiked sample. The explanation of this behaviour lies in the fact that spiked analytes coat the surface of natural or synthetic supports, so they may not be located on the same binding sites as native analytes. In addition, spiking techniques usually require an organic solvent as a vehicle to deposit the spiked analyte homogeneously onto the sample matrix, and this solvent may affect the chemical integrity of the sample and lead to deposition conditions different from native analytes. Competition between solvent molecules and analyte for strong adsorption sites when the analyte is deposited from a liquid solution may cause the analyte to be adsorbed on weak sites, thus allowing higher recoveries. Thus, a comparison between supercritical CO2 extraction of spiked and native PAHs from a synthetic matrix ( silica gel ) and from a soil sample revealed that the ef¢ciency from the natural sample was signi¢cantly lower ( mean yield of 41.78%) than that from silica ( mean recovery of 98.20%) [ 28 ], the addition of a polar modi¢er ( methanol ) being necessary in the former case. This different behaviour in the extraction of spiked versus natural analytes has permitted the study of the tightness of matrix^analyte binding. Such is the case with the study of the extraction kinetics of organic contaminants from environmental samples [ 29 ] in which it is shown that native compounds were extracted much more slowly than spiked ones. From these facts we can conclude that in deal-
ing with natural samples it is mandatory, in order to ensure quantitativeness, to determine the goodness of the SFE process by comparison with the performance of a Soxhlet extraction which is allowed to proceed exhaustively ( for 20 h ), with an arbitrary designation as 100% ef¢ciency [ 30 ]. In this sense, it is also relevant to take into account the fact that even Soxhlet cannot achieve a complete extraction, especially for analytes bound strongly to natural matrices. A recent report concerning the effectiveness of SFE of PAHs from natural matrix environmental samples [ 31 ] has led once again to the conclusion that the yield of extraction of speci¢c compounds from samples is dependent on the bulk composition of the sample ( matrix effects ). A consequence of the limited application of the technique to natural samples is the lack of standardised methods based on SFE. Only one SFE method has been adopted recently by the US EPA as an of¢cial method for the extraction of total petroleum hydrocarbons and PAHs from environmental matrices. In this sense, it is vital to perform in-depth studies on natural samples, as well as collaborative studies, with the aim of ¢nding a generic procedure ^ although this seems dif¢cult to establish.
3.3. Frequent need for clean-up As with other extraction methods, one of the problems involved in SFE is that the resulting extracts are not always free from unwanted matrix components, thus calling for clean-up steps. These steps are particularly necessary for fat-soluble analytes, while for environmental samples the extracts obtained using pure CO2 are frequently much cleaner than those obtained by solvent extraction, which can even make possible their direct injection onto the gas chromatograph. A number of sample clean-up methods ( selective adsorbents [ 32 ], immunoaf¢nity [ 33 ], etc. ) have been investigated for trapping the interfering substances. The trend in this matter is to integrate the clean-up step into the extraction cell in such a way that the sample cleanup can be done totally in the extractor. An alternative approach for avoiding the clean-up step, which includes the use of supercritical binary gas mixtures, has been proposed recently. A pressurised CO2 ^nitrogen binary £uid mixture has demonstrated suf¢cient solvating power for quantitative recoveries of traces of spiked organophosphorus and incurred organochlorine pesticides from poul-
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try fat. In addition, a signi¢cant reduction of lipid solubility in the extract was achieved, hence allowing the development of a method that produced an extract with a minimal lipid content, which could be used directly for gas chromatographic analysis, thereby eliminating the need for clean-up of the extract [ 34 ].
4. Conclusions Today, after an initial euphoric stage ( in the late 1980s and early 1990s ), SFE is a well-established and undoubtedly powerful technique in several areas, such as environmental, pharmaceutical, and polymer studies, and especially in food analysis, the last ( and particularly fat analysis ) being responsible for the current increase of supercritical £uid extractor sales [ 35 ]. Despite the technique's having the important limitations described above, especially for the extraction of polar analytes and the treatment of natural samples, the pretreatment of solid samples is undoubtedly accelerated by SFE with the help of special strategies for the extraction of medium polar and polar analytes. In order to establish where SFE is going it is mandatory to consider the latest contributions to this area. The coupling of supercritical CO2 and subcritical water is a very recent and promising alternative that has proved to extract non-polar analytes successfully. Thus, the coupling permits the class-selective fractionation of the relatively non-polar herbicide, Dacthal, from its very water-soluble metabolites in soil [ 36 ]. The development of new analyte collection methods, with reduced solvent consumption, which results in more concentrated extracts, is also a target of current research [ 37 ]. Another important future trend in SFE is the possibility of performing ¢eldwork. In this context, a single SFE method for ¢eld extraction of PCBs and PAHs in soils has been developed for use in combination with capillary GC^ECD or FID, without the need for clean-up steps after extraction [ 38 ]. Another portable system has been developed by the US Department of Defense and it is currently being tested. The approach uses dry ice ( thus avoiding compressed CO2 tanks ), and works well for the extraction of non-polar analytes, but requires the presence of modi¢ers for the extraction of nonpolar analytes. Taking into account the breakthroughs in other alternatives, such as accelerated solvent extraction or microwave-assisted Soxhlet,
we conclude that before selecting SFE as a primary extraction method, one should weigh its main bene¢ts ( cleanness, expeditiousness and simplicity ) and take into account its limitations. Thus, after considering the features of matrix and analytes, the analyst must select the most suitable technique from the competitive alternatives such as accelerated solvent extraction, microwave-assisted Soxhlet, and sub- and supercritical water extraction.
Acknowledgements Spain's Comisioèn Interministerial de Ciencia y Tecnolog|èa is gratefully thanked for ¢nancial support (Project PB95-1265 ).
References [ 1 ] M.D. Luque de Castro, M. Valcaèrcel, M.T. Tena, Analytical Supercritical Fluid Extraction, Springer-Verlag, Heidelberg, 1994. [ 2 ] E. Klesper, A.H. Corwin, D.A. Turner, J. Org. Chem. 27 ( 1962 ) 700. [ 3 ] T.L. Chester, J.D. Pinkston, D.E. Raynie, Anal. Chem. 70 ( 1998 ) 301R. [ 4 ] S.J. Letohay, J. Chromatogr. A 785 ( 1997 ) 289. [ 5 ] M. Valcaèrcel, M.T. Tena, Fresenius' J. Anal. Chem. 358 ( 1997 ) 369. [ 6 ] C.M. Wai, S. Wang, J. Chromatogr. A 785 ( 1997 ) 369. [ 7 ] J.R. Dean, S. Khunder, J. Pharm. Biomed. Anal. 15 ( 1997 ) 875. [ 8 ] K. Sugiyama, J. Chromatogr. A 332 ( 1985 ) 107. [ 9 ] R.W. Current, D.C. Tilotta, J. Chromatogr. A 785 ( 1997 ) 269. [ 10 ] R. Deuster, N. Lubahn, C. Friedrich, W. Kleiboehmer, J. Chromatogr. A 785 ( 1997 ) 227. [ 11 ] F.M. Lancas, M.S. Galhiane, S.R. Rissato, M.A. Barbirato, J. High Resolut. Chromatogr. 20 ( 1997 ) 369. [ 12 ] M.D. Burford, S.B. Hawthorne, D.J. Miller, Anal. Chem. 65 ( 1993 ) 1497. [ 13 ] M. Kane, J.R. Dean, S.M. Hitchen, C.J. Dowle, R.L. Tranter, Analyst 120 ( 1995 ) 355. [ 14 ] M.M. Jimeènez-Carmona, M.T. Tena, M.D. Luque de Castro, J. Chromatogr. A 711 ( 1995 ) 269. [ 15 ] M.D. Luque de Castro, M.T. Tena, Trends Anal. Chem. 15 ( 1996 ) 32. [ 16 ] M.D. Luque de Castro, M.M. Jimeènez-Carmona, Trends Anal. Chem. 17 ( 1998 ) 441. [ 17 ] S.B. Hawthorne, Y. Yang, D.J. Miller, Anal. Chem. 6 ( 1994 ) 2912. [ 18 ] Y. Yang, S.B. Hawthorne, D.J. Miller, Environ. Sci. Technol. 31 ( 1997 ) 430. [ 19 ] M.M. Jimeènez-Carmona, J.J. Mancluès, A. Montoya, M.D. Luque de Castro, J. Chromatogr. 785 ( 1997 ) 329. [ 20 ] A. Basile, M.M. Jimeènez-Carmona, A.A. Clifford, J. Food Agric. Chem. 46 ( 1998 ) 5205.
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[ 21 ] M.M. Jimeènez-Carmona, J.L. Ubera, M.D. Luque de Castro, J. Chromatogr. A 855 ( 1999 ) 625. [ 22 ] Y. Yang, A. Garaibeth, S.B. Hawthorne, D.J. Miller, Anal. Chem. 67 ( 1995 ) 641. [ 23 ] J.A. Field, D.J. Miller, T.M. Field, S.B. Hawthorne, W. Giger, Anal. Chem. 64 ( 1992 ) 3161. [ 24 ] S.B. Hawthorne, D.J. Miller, D.E. Nievens, D.C. White, Anal. Chem. 64 ( 1992 ) 405. [ 25 ] Y. Cai, R. Alzaga, J.M. Bayona, Anal. Chem. 66 ( 1994 ) 1161. [ 26 ] Y. Liu, V. Loèpez-Aèvila, M. Alcaraz, Anal. Chem. 66 ( 1994 ) 3788. [ 27 ] M.M. Jimeènez-Carmona, M.D. Luque de Castro, Anal. Chem. 70 ( 1998 ) 2100. [ 28 ] M.T. Tena, M.D. Luque de Castro, M. Valcaèrcel, Chromatographia 38 ( 1994 ) 431. [ 29 ] N. Alexandrou, J. Pawliszyn, Anal. Chem. 61 ( 1989 ) 2770.
[ 30 ] B.A. Benner Jr., Anal. Chem. 70 ( 1998 ) 4594. [ 31 ] N. Alexandrou, M.J. Lawrence, J. Pawliszyn, Anal. Chem. 64 ( 1992 ) 301. [ 32 ] V. Pinchon, E. Aulard-Macler, H. Oubihi, M.C. Hennion, M. Caude, Chromatographia 46 ( 1997 ) 529. [ 33 ] J.W. King, Z. Zhang, Anal. Chem. 70 ( 1998 ) 1431. [ 34 ] Anal. Chem. News Features 70 ( 1998 ) 333A. [ 35 ] J.A. Field, K. Monohan, R. Reed, Anal. Chem. 70 ( 1998 ) 1956. [ 36 ] J. Vejrosta, P. Karaèsebe, J. Planeta, Anal. Chem. 71 ( 1999 ) 905. [ 37 ] J.J. Langenfeld, S.B. Hawthorne, D.J. Miller, J. Pawliszyn, Anal. Chem. 67 ( 1995 ) 1727. [ 38 ] S. BÖwadt, L. Mazeas, D.J. Miller, S.B. Hawthorne, J. Chromatogr. A 785 ( 1997 ) 205.
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Where is supercritical £uid extraction going? M.D. Luque de Castro*, M.M. Jimeènez-Carmona
Analytical Chemistry Division, Faculty of Sciences, E-14004 Coèrdoba, Spain The status of supercritical £uid extraction as an analytical technique for treatment of solid samples is discussed. Its most signi¢cant advantages are its preconcentration effect, cleanness and safety, quantitativeness, expeditiousness and simplicity. Its limitations include the dif¢culty of extracting polar analytes, owing to the non-polar character of the CO2 which is used, the different recoveries obtained from spiked and natural samples, and the frequent need for clean-up steps after extraction. A critical discussion is given of these factors, as well as possible solutions and new alternatives such as the use of sub- and supercritical water and accelerated solvent extraction. z2000 Elsevier Science B.V. All rights reserved. Keywords: Analytical supercritical £uid extraction; Sample treatment; Polar analyte
1. Introduction The continued search for rapid, ef¢cient and cost-effective means of sample treatment has introduced supercritical £uids into analytical chemistry, giving us supercritical £uid chromatography (SFC ) and supercritical £uid extraction (SFE ) [ 1 ]. Both techniques seek to exploit the unique properties of gases at temperatures and pressures above the critical point. SFE is a somewhat atypical technique in its chronological evolution. Its chromatographic counterpart, SFC, was developed at an earlier stage ( ¢rst demonstrated in 1962 [ 2 ]), which is in clear contrast with other extraction techniques whose batch modes were developed much earlier than their continuous modes ( e.g., liquid^liquid extraction ). On the other hand, the fairly extensive commercial availability of supercritical £uid extractors when the technique was introduced was inconsistent with the relative scarcity of associated publications and developments, as compared to most analytical *Corresponding author. Fax: +34 (957) 218 606. E-mail: [email protected]
techniques. Traditionally, the manufacture of commercially available apparatus and instruments, which has fostered the development of new applications and their customary use, has followed scienti¢c and technical advances and feasibility tests. These seemingly contradictory facts are easy to explain, though, in that SFE aids ef¢cient development of the ¢rst stage of the analytical process, which is also the most needed in research. The SFE literature shows the predominant use of CO2 as extractant ( more than 98% of the applications have been developed using this £uid ) and calls for an inspection of its features to show whether the decreased research in this ¢eld is related to the extractant itself. In the authors' opinion, supercritical CO2 has signi¢cant advantages, but also shows important limitations that can be overcome with the help of new techniques and analytical approaches. An ideal extraction method should be rapid, simple, and inexpensive to perform. It should yield quantitative recovery of the target analytes without loss or degradation; provide a sample that is immediately ready for analysis without additional concentration or class fractionation steps; and generate no additional laboratory wastes. Unfortunately, liquid solvent extractions frequently fail to meet these goals. They often require several hours ^ or even days ^ to perform, result in a dilute extract ( which must be concentrated when trace analysis is required ), and may not result in quantitative recovery of the target analytes. Recent concerns about the hazardous nature of many commonly used solvents, the costs and environmental dangers of waste solvent disposal, and the emission of hazardous solvents into the atmosphere, have led to the development of alternative extraction methods. In this context, SFE ^ the technique based on the use of supercritical £uids as leaching agents ^ emerged in the mid-1980s as a promising tool to overcome the dif¢culties of solid sample extraction. The majority of analytical supercritical £uid extractions have used supercritical CO2 as extracting agent because of its preferred critical properties ( critical temperature and pressure, 31³C and 72 bar, respec-
0165-9936/00/$ ^ see front matter PII: S 0 1 6 5 - 9 9 3 6 ( 9 9 ) 0 0 2 2 8 - 9
ß 2000 Elsevier Science B.V. All rights reserved.
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trends in analytical chemistry, vol. 19, no. 4, 2000
tively ), low toxicity, and chemical inertness. Despite the promising features of this technique, it has not ful¢lled the expectations of researchers so far. Thus, many analysts were quick to try the technique but became frustrated and gave up SFE systems that did not live up to their expectations, leading to a reduction in the number of SFE instrument manufacturers. Here we review critically the current state of SFE in analytical chemistry, considering all aspects of the technique, as well as solutions to the drawbacks, and the foreseeable trends. Other reviews on this issue have been published recently, referring to general aspects of the technique [ 3 ], and speci¢c matters such as the use of SFE in food analysis [ 4,5 ], in the extraction of metals as complexes [ 6 ], or in the extraction of pharmaceuticals [ 7 ].
2. Advantages of SFE The bene¢ts of SFE ( focused on the use of supercritical CO2 , since this is mainly used ), versus conventional techniques which are based on the rapid diffusion of the analytes in the £uid ( gas-like diffusion ) and the £uid solvation power ( liquid-like solvation ), have led to SFE being proposed as an alternative to conventional liquid solvent extractions such as Soxhlet and ultrasonic extractions. The most signi¢cant advantages of the SFE technique are the preconcentration effect it has, its cleanness and safety, its quantitativeness, expeditiousness, and its simplicity and selectivity.
2.1. Preconcentration effect The preconcentration effect allowed by CO2 -SFE is one of its most signi¢cant advantages versus other extraction techniques, since the large solvent volumes used in Soxhlet extractions ( 50^200 ml ) must be concentrated to 2^5 ml by using an appropriate device prior to analysis in order to obtain adequate analyte concentrations. This step usually takes between 2 and 24 h, and releases substantial amounts of solvents into the atmosphere. By contrast, SF extracts are usually collected in smaller volumes ( 10^20 ml maximum ), thus avoiding the preconcentration steps required after other conventional techniques which are commonly used for sample extraction or digestion. The solvent changeover occasionally required for the isolation or determination of analytes is
most readily done in SFE. After extraction, the SF is separated by depressurisation and the analytes are collected by bubbling them through a suitable solvent, or deposited on a sorbent or impact solid, and subsequently dissolved in the most appropriate solvent for the intended purpose.
2.2. Cleanness and safety Extraction by SC-CO2 is cleaner and less hazardous than conventional extraction since CO2 is nontoxic and non-£ammable and, unlike toxic solvents ( n-hexane, ether, methylene chloride ), carbon dioxide poses no ¢re risk and leaves no environmentally hazardous wastes.
2.3. Quantitativeness Conventional extraction methods do not necessarily rely on complete isolation of the analyte, so increasing the recovery of an analyte, or extracting it quantitatively, detracts from the extraction's precision and reliability. The SFE extraction of 2,3,7,8tetrachlorodibenzo-p-dioxin from sediments [ 8 ] is a good example of how quantitative the extraction with SFs can be. Virtually 100% of the analyte was extracted within 30 min from the sediment, whilst Soxhlet extraction of the same sample for 18 h provided only 65% recovery. The recovery achieved by SFE ( under optimum working conditions ) can sometimes be even higher than that provided by Soxhlet. This is because the energy provided by solvents in Soxhlet extraction is not high enough to break the very strong bindings that sometimes occur with natural samples, and can necessitate the use of more drastic conditions such as those provided by SFE. This is the case with the extraction of hydrocarbons [ 9 ] or nitroaromatic and polyaromatic compounds [ 10 ] from soils. The solvent power, polarity, and working temperatures of the SFE, as well as its high diffusivity, are the chief factors on which complete extraction relies.
2.4. Expeditiousness One of the most valuable properties of SFE relative to classic separation techniques is expeditiousness. In fact, it is faster than conventional liquid extraction since: ( a ) the SFE penetrates into solid matrices more rapidly than do liquid solvents; ( b ) no concentration is required after extraction; and ( c ) the time needed to make the sample ready for
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measurement is up to 100 times shorter in many cases. Thus, CO2 extraction at 300 atm during 5 min gave recoveries of diclofol residues from ¢sh slightly higher ( 94.1^99.8%) than those provided by Soxhlet extraction with hexane during 8 h ( 87.9^92.3%) [ 11 ].
2.5. Simplicity SFE involves a very small number of steps, so it leads to short analysis times and reduced sample transfer, and hence to small analytical errors. The simplicity of SFE is clearly re£ected in the extraction of atrazine from soil samples. Supercritical CO2 extraction of a soil sample for 15 min with no previous treatment is enough to obtain recoveries comparable to those provided by Soxhlet extraction after sample pretreatment.
2.6. Selectivity One of the most important advantages of SFE versus conventional extraction techniques is its selectivity. Thus, SFE allows selective extraction of a wide range of analytes (PAHs, PCBs, and organochlorides ) [ 12 ] while not extracting the bulk matrix ( e.g., humic acids ). This behaviour allows the extract to be ready for direct analysis without additional manipulation.
3. Limitations of SFE Supercritical CO2 extraction, whose most important bene¢ts have been described, also shows signi¢cant limitations that are worth studying in order to know the ¢eld of application of the technique and help one seek for alternatives in this context. The main drawbacks SFE has to cope with are the dif¢culties in extracting polar analytes, the different ef¢ciencies obtained from spiked and natural samples, and the need for a clean-up step prior to measurement.
3.1. Dif¢culties in extracting polar analytes As stated above, the most popular £uid for SFE has been CO2 because of its low critical properties, low toxicity, and chemical inertness. Although CO2 is an excellent solvent for non-polar analytes, its main shortcoming is its low dielectric constant, which makes the extraction of polar and ionic com-
pounds dif¢cult. As examples, analytes such as alcohol phenol ethoxylate [ 13 ] and clenbuterol [ 14 ] are not extracted at all when pure CO2 is used as extractant. The strategies used in this context for facilitating the SFE of polar and ionic compounds have been reviewed recently [ 15 ] and are based on two general principles. The ¢rst strategy is to increase the polarity of the CO2 used as extractant, by changing the physical parameters of the supercritical CO2 , or by using supercritical £uids other than CO2 . The ¢rst choice would be a £uid with higher solvent strength, but the use of more polar £uids is often limited by practical considerations. Supercritical ammonia would be very attractive from a solvent strength standpoint, but it is dif¢cult to pump ( it tends to dissolve pump seals ), is chemically reactive, and is too dangerous for routine use. Supercritical methanol is also an excellent solvent but is less attractive because of its high critical temperature and because it is a liquid under ambient conditions, which complicates sample concentration after extraction. One of the most promising alternatives in this context is sub- or supercritical water, whose potential for extraction has been reviewed recently [ 16 ]. The most outstanding feature of this leaching agent is the possibility of manipulating its dielectric constant, making possible a drastic reduction in this parameter by raising the temperature under moderate pressure. For example, the dielectric constant of water decreases from 80 at ambient pressure and temperature, to 27 at 250³C and pressure s 40 atm, to a value below 10 under supercritical conditions. Thus, water has been used successfully to extract from environmental samples pollutants with a wide range of polarities, by sequentially raising the temperature [ 17,18 ], as well as for the extraction of pesticides from soils [ 19 ] and of essential oils from natural products [ 20,21 ]. Another way of increasing the polarity of the CO2 is by the addition of co-solvents, mainly methanol [ 22 ]. Modi¢ers can be introduced as mixed £uids in the pumping system, or with the aid of a second pump, or by simply injecting the modi¢er as a liquid onto the sample before beginning the extraction. A second group of strategies used for enhancing the SFE of polar and ionic compounds is based on reducing the polarity of the analyte to be extracted, by ion pair formation [ 23 ], esteri¢cation, or analogous reactions [ 24 ], organometallic compound formation [ 25 ], complex formation [ 26 ], or reverse micelle formation [ 27 ]. All these strategies compli-
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cate the experimental set-up and / or sample handling.
3.2. Dif¢culties in dealing with natural samples The samples typically subjected to SFE are solids, although the starting sample can be also semi-solid, liquid or gaseous. When the sample is not solid, it must be retained on an appropriate support ( a natural matrix from which the target analytes are removed previously, or a synthetic support such as glass wool or a diatomaceous earth ) in order to ensure effective attack by the supercritical £uid. It is also worth noting that most research work on SFE has been conducted on spiked samples. One of the most signi¢cant limitations of the technique in this context is that comparative studies involving natural and spiked samples have shown the former to deviate from the foreseeable behaviour in that the analytes bind more strongly in natural samples, which makes unfeasible the prediction of the behaviour of a natural sample from the results provided by a spiked sample. The explanation of this behaviour lies in the fact that spiked analytes coat the surface of natural or synthetic supports, so they may not be located on the same binding sites as native analytes. In addition, spiking techniques usually require an organic solvent as a vehicle to deposit the spiked analyte homogeneously onto the sample matrix, and this solvent may affect the chemical integrity of the sample and lead to deposition conditions different from native analytes. Competition between solvent molecules and analyte for strong adsorption sites when the analyte is deposited from a liquid solution may cause the analyte to be adsorbed on weak sites, thus allowing higher recoveries. Thus, a comparison between supercritical CO2 extraction of spiked and native PAHs from a synthetic matrix ( silica gel ) and from a soil sample revealed that the ef¢ciency from the natural sample was signi¢cantly lower ( mean yield of 41.78%) than that from silica ( mean recovery of 98.20%) [ 28 ], the addition of a polar modi¢er ( methanol ) being necessary in the former case. This different behaviour in the extraction of spiked versus natural analytes has permitted the study of the tightness of matrix^analyte binding. Such is the case with the study of the extraction kinetics of organic contaminants from environmental samples [ 29 ] in which it is shown that native compounds were extracted much more slowly than spiked ones. From these facts we can conclude that in deal-
ing with natural samples it is mandatory, in order to ensure quantitativeness, to determine the goodness of the SFE process by comparison with the performance of a Soxhlet extraction which is allowed to proceed exhaustively ( for 20 h ), with an arbitrary designation as 100% ef¢ciency [ 30 ]. In this sense, it is also relevant to take into account the fact that even Soxhlet cannot achieve a complete extraction, especially for analytes bound strongly to natural matrices. A recent report concerning the effectiveness of SFE of PAHs from natural matrix environmental samples [ 31 ] has led once again to the conclusion that the yield of extraction of speci¢c compounds from samples is dependent on the bulk composition of the sample ( matrix effects ). A consequence of the limited application of the technique to natural samples is the lack of standardised methods based on SFE. Only one SFE method has been adopted recently by the US EPA as an of¢cial method for the extraction of total petroleum hydrocarbons and PAHs from environmental matrices. In this sense, it is vital to perform in-depth studies on natural samples, as well as collaborative studies, with the aim of ¢nding a generic procedure ^ although this seems dif¢cult to establish.
3.3. Frequent need for clean-up As with other extraction methods, one of the problems involved in SFE is that the resulting extracts are not always free from unwanted matrix components, thus calling for clean-up steps. These steps are particularly necessary for fat-soluble analytes, while for environmental samples the extracts obtained using pure CO2 are frequently much cleaner than those obtained by solvent extraction, which can even make possible their direct injection onto the gas chromatograph. A number of sample clean-up methods ( selective adsorbents [ 32 ], immunoaf¢nity [ 33 ], etc. ) have been investigated for trapping the interfering substances. The trend in this matter is to integrate the clean-up step into the extraction cell in such a way that the sample cleanup can be done totally in the extractor. An alternative approach for avoiding the clean-up step, which includes the use of supercritical binary gas mixtures, has been proposed recently. A pressurised CO2 ^nitrogen binary £uid mixture has demonstrated suf¢cient solvating power for quantitative recoveries of traces of spiked organophosphorus and incurred organochlorine pesticides from poul-
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try fat. In addition, a signi¢cant reduction of lipid solubility in the extract was achieved, hence allowing the development of a method that produced an extract with a minimal lipid content, which could be used directly for gas chromatographic analysis, thereby eliminating the need for clean-up of the extract [ 34 ].
4. Conclusions Today, after an initial euphoric stage ( in the late 1980s and early 1990s ), SFE is a well-established and undoubtedly powerful technique in several areas, such as environmental, pharmaceutical, and polymer studies, and especially in food analysis, the last ( and particularly fat analysis ) being responsible for the current increase of supercritical £uid extractor sales [ 35 ]. Despite the technique's having the important limitations described above, especially for the extraction of polar analytes and the treatment of natural samples, the pretreatment of solid samples is undoubtedly accelerated by SFE with the help of special strategies for the extraction of medium polar and polar analytes. In order to establish where SFE is going it is mandatory to consider the latest contributions to this area. The coupling of supercritical CO2 and subcritical water is a very recent and promising alternative that has proved to extract non-polar analytes successfully. Thus, the coupling permits the class-selective fractionation of the relatively non-polar herbicide, Dacthal, from its very water-soluble metabolites in soil [ 36 ]. The development of new analyte collection methods, with reduced solvent consumption, which results in more concentrated extracts, is also a target of current research [ 37 ]. Another important future trend in SFE is the possibility of performing ¢eldwork. In this context, a single SFE method for ¢eld extraction of PCBs and PAHs in soils has been developed for use in combination with capillary GC^ECD or FID, without the need for clean-up steps after extraction [ 38 ]. Another portable system has been developed by the US Department of Defense and it is currently being tested. The approach uses dry ice ( thus avoiding compressed CO2 tanks ), and works well for the extraction of non-polar analytes, but requires the presence of modi¢ers for the extraction of nonpolar analytes. Taking into account the breakthroughs in other alternatives, such as accelerated solvent extraction or microwave-assisted Soxhlet,
we conclude that before selecting SFE as a primary extraction method, one should weigh its main bene¢ts ( cleanness, expeditiousness and simplicity ) and take into account its limitations. Thus, after considering the features of matrix and analytes, the analyst must select the most suitable technique from the competitive alternatives such as accelerated solvent extraction, microwave-assisted Soxhlet, and sub- and supercritical water extraction.
Acknowledgements Spain's Comisioèn Interministerial de Ciencia y Tecnolog|èa is gratefully thanked for ¢nancial support (Project PB95-1265 ).
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